PROJECT SUMMARY: Neuroblastoma (NB), a predominantly pediatric cancer of neural crest cell origin, is a clinically challenging disease due to the genomic and intratumoral heterogeneity. There are three risk stratifications to characterize the disease - low-risk, intermediate-risk, and high-risk - which are based on several factors such as mutation burden and age at diagnosis. High-risk NB patients have a poor prognosis, with a 5- year survival rate of roughly 50% despite dose-intensive chemoradioimmunotherapy treatment. The few common genomic aberrations in high-risk NB include amplification of the MYCN oncogene in 40% of cases, aberrations in the receptor tyrosine kinase (RTK) Anaplastic Lymphoma Kinase (ALK) in 14% of cases such as ALK gene amplification and three hot spot mutations at the F1174, F1245, and R1275 residues conferring ligand- independent kinase activity, and mutations in the PTPN11 gene, which encodes the SHP2 protein, in 3.4% of cases. The only targeted therapy for high-risk NB treatment is ALK inhibition, and despite the fact that the third generation ALK inhibitor lorlatinib is more potent and superior than previous generation ALK inhibitors, relapse in patients on lorlatinib occurs. Thus, the discovery of new targeted therapies and dosing schedules to overcome the challenges endured in the clinic is paramount. A novel therapeutic drug target, the Src homology 2-containing tyrosine phosphatase (SHP2), is a non-receptor protein tyrosine phosphatase implicated in several disease states including Noonan Syndrome, LEOPARD Syndrome, and a variety of cancers. SHP2 plays a critical role in the full activation of the RAS/MAPK signaling cascade, as well as signal transduction from several RTKs to downstream pathways. Research aimed at understanding the role of SHP2 has largely been focused on the catalytic function with respect to signal transduction. I hypothesize that SHP2 is a tractable therapeutic vulnerability in ALK-driven high-risk NB. Recently, an allosteric small molecule inhibitor which targets SHP2, SHP099, has been discovered. While our preliminary data show that inhibiting SHP2 catalytic activity via SHP099 is not sufficient to prevent NB tumor cell viability, recent studies reveal that SHP2 inhibition shows translational promise in combination with other targeted therapies such as ALK inhibitors. It is imperative to define the functional effect of the SHP2 mutants observed in NB due to the mutations in PTPN11. Therefore, I will characterize the mutations in PTPN11 and investigate the phosphatase-independent role of SHP2 in high-risk NB. These studies will shed light on this overlooked potential role of SHP2 and allow for more rational drug development targeting SHP2 to result in a clinically impactful treatment option. In addition to defining the functional role of SHP2, I will study the combination of lorlatinib and SHP099 to determine anti-tumor activity and survival of ALK-driven high-risk NB upon combination treatment. These studies will provide evidence for which patient populations would benefit from this combination for future clinical trial design and translation to the clinic.